Relatively little is known about how myofibrils are assembled, or how the myofibrils are maintained in the face of repeated muscle activity. This proposal outlines studies of three genes in C. elegans, unc-98, unc-96 and unc-97, which are required for proper myofibril organization. UNC-98 is a novel 310 residue polypeptide consisting of four C2H2 Zn fingers and predicted NLS and NES sequences. By use of UNC-98 antibodies and UNC-98::GFP fusions, UNC-98 resides at M-lines, muscle cell nuclei, and probably at dense bodies (Z line analogs). By 2-hybrid experiments, UNC-98 interacts with UNC-97 (PINCH in mammals), a LIM domain protein required for worm muscle focal adhesion assembly. Like UNC-98, UNC-97::GFP had been shown to localize to muscle dense bodies, M-lines and nuclei. It is hypothesized that UNC-98 and UNC-97 function in muscle focal adhesion homeostasis, in which these proteins when localized to the adhesion sites monitor muscle activity, and travel to the nucleus to affect transcription. Because of their similar mutant phenotypes, genetic interaction, and requirement of unc-96 activity for proper UNC-98 localization, it is hypothesized that UNC-96 and UNC-98 interact or work together. Goals include: (1) study UNC-98 including mapping regions required for nuclear vs. attachment structure localization, to determine whether, UNC-98 moves from myofibrils to the nucleus, to characterize new unc-98 mutant alleles, to characterize several suspected partners and to identify new partners for UNC-98, and to determine whether UNC-98 influences the expression of other genes; (2) to determine the nature of the UNC-96 protein, its intracellular location and molecular partners; and (3) to study UNC-97 including to determine whether UNC-97 moves from myofibrils to nucleus, whether nuclear localization depends on UNC-98, localize UNC-97 with antibodies, seek further evidence for UNC-97's interaction with several known and new proteins, isolate genetic modifiers of unc-97,and to determine whether UNC-97 influences the expression of other genes. ? ?
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